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Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast

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Abstract

The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-l-arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-l-arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

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Acknowledgments

We thank Dr. Michael Hahn (CCRC, UGA) for the pBAt plasmid and Adam Bogdanove (ISU) for genomic DNA of X. oryzae. We are grateful to Andre Salazar (BBMB, ISU) for his valuable technical assistance. This work was supported by the grant #09-3384 obtained from Roy J. Carver Charitable Trust (2009–2011) and we acknowledge their support.

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Authors and Affiliations

  1. Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 3212 MBB, Ames, IA, 50011, USA

    Gennady Pogorelko, Oksana Fursova, Ming Lin, Eric Pyle, Johanna Jass & Olga A. Zabotina

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  1. Gennady Pogorelko
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  2. Oksana Fursova
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  3. Ming Lin
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  4. Eric Pyle
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  5. Johanna Jass
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  6. Olga A. Zabotina
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Correspondence to Olga A. Zabotina.

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Gennady Pogorelko, Oksana Fursova and Ming Lin contributed equally.

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Pogorelko, G., Fursova, O., Lin, M. et al. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast. Plant Mol Biol 77, 433 (2011). https://doi.org/10.1007/s11103-011-9822-9

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